<li>Right now you'll be in viewing mode, which lets you rotate and move the camera. To switch to "Editing mode" click on the box in the bottom right that reads "3-button viewing mode". This will switch you to editing mode.

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<li>Here you'll have all the options for actually moving your protein around and rotating it.

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<li>On the PYmol menu and script window, you can switch to "Builder". This will give you specific options for adding amino acids and other molecules to your structure.

+

<li>To make an insertion, you'll have to delete a bond. This is one of the options in the Builder window. Or you can delete a residue or residues, by selecting a residue in viewer mode, right clicking and choosing remove.

+

<li>Now you have free bonds where you can build residues. Simply click on the atom where you want to add a residue (usually a terminal N or C=O), then click on the residue you want to add in the Builder window. The default secondary structure is in a parallel beta sheet, which is essentially just a straight line.

+

<li>Rotating bonds can be done by clicking on an atom and then double-click and hold the part that you want to rotate. Remember, don't rotate between N and C=O bonds!

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<li>The last and most important thing you'll need to know, which isn't obvious at first is how Pymol actually make bonds. It does it purely by proximity. So if you have atoms that are within 1.5 angstroms, Pymol will automatically make a bond between those two atoms after you save and reload the .pdb file.

+

<li>The code "distance (pk1),(pk2)" will measure the distance between two selected atoms. You may also use the measurement wizard to find distances and well as phi and psi angles.

+

<li>Move and twist your bonds and proteins to build your chimeric protein and then save them as one .pdb file!

This is something that we are just getting into. Basically, what GROMACS does is simulates your protein structure or other molecule floating in a solvent (usually water). This can be really useful in estimating how your protein will actually behave with regards to its stability and flexibility. After running this you'll be able to see whether or not your enzyme's catalytic site might be facing the wrong way!

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Below is a link to a script, which contains a breakdown of each variable and how to actually do the run.

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<a href="http://2012.igem.org/wiki/images/e/e6/Run_md_gromos_cutoff.txt">Click here!</a> and then rename the script to a .sh instead of .txt.

In our chimeras we have linker sequences on either side of the chimeric insert sequence. The linkers we chose to use were inert, and flexible linker sequences, with different sequences on either side so that overlap extension will be successful.

FliC -GGGGS- insert -GGSGG- FliC

It is important to also consider the flexible regions of the protein in which you are making your insertion. Flexible regions may be able to accommodate a protein insertion, and would not require very long linkers. However, longer linkers may be required to introduce enough flexibility for the inserted protein to fold properly.

Structures

The protein database is possibly the most important tool in chimeric protein design. Without having any previous knowledge of your structure, it is impossible to really say where or how you would design your chimeric protein.
In our case, the crystal structure for E. coli flagellin is not available on the protein database. However, the structure for the flagellin of S. typhymurium was available and is very similar to the structure of E. coli flagellin. The PDB ID is 1UCU.

Additionally, some general assumptions can be made when searching for a good place to make an insertion. For example, the site that we chose to make our variable domain insertions is:

...AVTVANDGTVTMATG...

The insertion was made in between AVT and TVT, replacing the amino acid sequence VANDG. We needed to have some spacing in between the overlap regions for PCR overlap extension, which is why VANDG was replaced.

The presence of several threonine, alanine, valine and glycine residues is indicative of a loop region, which would make a good spot for an insertion. Additionally, when we used PCR to make the overlapping regions for the insertion, we amplified off of the already existing linker used for ovarlapping as a deletion. Hence our total linker was:

Right now you'll be in viewing mode, which lets you rotate and move the camera. To switch to "Editing mode" click on the box in the bottom right that reads "3-button viewing mode". This will switch you to editing mode.

Here you'll have all the options for actually moving your protein around and rotating it.

On the PYmol menu and script window, you can switch to "Builder". This will give you specific options for adding amino acids and other molecules to your structure.

To make an insertion, you'll have to delete a bond. This is one of the options in the Builder window. Or you can delete a residue or residues, by selecting a residue in viewer mode, right clicking and choosing remove.

Now you have free bonds where you can build residues. Simply click on the atom where you want to add a residue (usually a terminal N or C=O), then click on the residue you want to add in the Builder window. The default secondary structure is in a parallel beta sheet, which is essentially just a straight line.

Rotating bonds can be done by clicking on an atom and then double-click and hold the part that you want to rotate. Remember, don't rotate between N and C=O bonds!

The last and most important thing you'll need to know, which isn't obvious at first is how Pymol actually make bonds. It does it purely by proximity. So if you have atoms that are within 1.5 angstroms, Pymol will automatically make a bond between those two atoms after you save and reload the .pdb file.

The code "distance (pk1),(pk2)" will measure the distance between two selected atoms. You may also use the measurement wizard to find distances and well as phi and psi angles.

Move and twist your bonds and proteins to build your chimeric protein and then save them as one .pdb file!

GROMACS

This is something that we are just getting into. Basically, what GROMACS does is simulates your protein structure or other molecule floating in a solvent (usually water). This can be really useful in estimating how your protein will actually behave with regards to its stability and flexibility. After running this you'll be able to see whether or not your enzyme's catalytic site might be facing the wrong way!
Below is a link to a script, which contains a breakdown of each variable and how to actually do the run.
Click here! and then rename the script to a .sh instead of .txt.